Antigen binding formats for use in therapeutic treatments or diagnostic assays

ABSTRACT

The present invention relates to antigen binding formats for use in therapeutic treatments or diagnostic assays. The present invention relates to an antigen-binding format consisting of:—a first fusion protein wherein the CH1 constant domain of an antibody is fused i) by its N-terminal end to the C-terminal end of a variable domain of an antibody and ii) by its C-terminal end to the N-terminal end of a variable domain of an antibody and,—a second fusion protein wherein the CL constant domain of an antibody is fused by its N-terminal end to the C-terminal end of a variable domain of an antibody.

FIELD OF THE INVENTION

The present invention relates to antigen binding formats for use intherapeutic treatments or diagnostic assays.

BACKGROUND OF THE INVENTION

Therapeutic monoclonal antibodies (mAbs) have met some successes in theclinic over the last years, particularly in oncology. More than twentyfive mAbs are on the market.

Many technical efforts have been made to generate second generation mAbswith decreased immunogenicity and with optimized effector functions.Since the majority of therapeutic antibodies are IgG1, at least part ofthe observed in vivo effects of mAbs might be induced followinginteractions between their Fc region and FcγR. Notably, the ability ofmAbs to kill tumor cells has been related to their capacity to recruitand activate effector cells such as NK cells and macrophages throughreceptors for the Fc portion of IgG (FcγR).

However, recent reports have shown that the efficiency of IgG1 humantherapeutic mAbs is strongly affected by various parameters: changes inFc glycosylation, FcγRIIIA polymorphism, interaction with inhibitoryFcγRIIB, and competition with endogenous IgG for FcγRI and FcγRIIIbinding. For instance, studies with FcγR−/− mice have revealed theimplication of different FcγR in some in vivo mechanisms of action oftwo widely used therapeutic mAbs, trastuzumab and rituximab. Thesecytotoxic mAbs directed against tumors engage both activating (FcγRIIIA)and inhibitory (FcγRIIB) receptors. In these studies, a more pronouncedtumor regression was observed in FcγRIIB-deficient mice than inwild-type mice, whereas FcγRIIIA-deficient mice were unable to stoptumor growth in the presence of therapeutic mAbs. In humans, a recentstudy has shown that the therapeutic efficiency of rituximab (a chimerichuman IgG1) in patients with non-Hodgkin lymphoma is partly correlatedwith FcγRIIIA polymorphism. Thus, patients homozygous for the Val158allele (IgG1 high binder) exhibited a higher response to the treatmentthan the patients homozygous for the Phe158 allele (IgG1 low binder).Similarly, engineered IgG glycoforms have been shown to triggeroptimized ADCC through the recruitment of FcγRIIIA. A first study showedthat an IgG1 antibody engineered to contain increasing amounts ofbisected complex oligosaccharides (bisecting N-acetylglucosamine,GlcNAC) allows the triggering of a strong ADCC as compared to itsparental counterpart. Second, a lack of fucose or low fucose content onhuman IgG1 N-linked oligosaccharides has been shown to improve FcγRIIIAbinding and ADCC as well as to increase the clearance rate of Rhesus D+red blood cells in human volunteers. Moreover, it has been recentlyshown that the antigenic density required to induce an efficient ADCC islower when the IgG has a low content in fucose as compared to a highlyfucosylated IgG.

The idea that a better control of Fc/FcγR interactions was needed whenusing therapeutic mAbs has been clearly argued in the early 80's and ledto the concept and the generation of bispecific antibodies (bsAbs),using biochemical approaches and then molecular engineering in the early90's. Bispecific antibodies, able to bring together target cells andactivated effector cells have important potential advantages over wholenaked mAbs. Notably, with regard to NK cells recruitment and activation,bsAbs make it possible to overcome most of the problems encountered withtherapeutic mAbs. First, it is far easier to use an antibody arm bindingto FcγRIIIA than to engineer and fine-tune the interaction between theantibody Fc region and FcγRIIIA. It is indeed possible to select aFcγRIIIA binder devoid of cross reaction for inhibitory FcγRIIB andtargeting an epitope not involved in the Fc binding to avoid thehigh/low binder FcγRIIIA polymorphism issue, as well as endogenous IgGcompetition. Moreover, antibody fragments are not concerned byglycosylation issues, and it is possible to fine-tune the affinity ofthe antibody from a μM to a nM range, i.e., an affinity up to 1,000 foldhigher than that involved in Fc/FcγRIIIA interaction. Thus, a number ofattempts have been made to create anti-FcγRIIIA×anti-target bsAbs.

However, for years, these attempts were hindered by the impossibility toefficiently produce such molecules, the most efficient techniquesrequiring grams of mAbs to produce milligrams of heterogeneouspreparations of bsAbs. Therefore, the first generation of bsAbs neverreached the market, mostly due to the cost of getting molecules withbi-functional properties in large amounts for a therapeutic use.

The inventors have recently developed a new generation of bispecificantibodies, based on llama VHH (sdAb for single domain antibody or alsoNb for nanobody), that can be easily produced in E. coli and thatovercome the limitations listed above, while being able to exert astrong tumor lysis at extremely low concentrations. These bispecificantibodies are described in the International Patent Application noWO/2006/064136. This generation of therapeutic antibodies has thepotential to rapidly translate into efficient therapeutics. Althoughthese bsAbs accumulate within the tumor, they suffer from a rapidelimination due to their relatively small size, below the renalthreshold (around 60 kDa), and to the absence of Fc region involved inthe interaction with the FcRn receptor, responsible for the long serumhalf-life of full length IgG. Thus, there is a need to improve thesebsAbs in terms of efficiency, serum half-life and biodistribution.

SUMMARY OF THE INVENTION

The present invention relates to an antigen-binding format consistingof:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a first variable domain of an antibody and ii) by its        C-terminal end to the N-terminal end of a second variable domain        of an antibody and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a third variable domain of an antibody.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have now created new antigen binding formats to increasethe serum half-life of the original bsAb format (54 kDa) described inthe International Patent Application WO/2006/064136 by fusing oneadditional sdAb (single domain antibody) to the C-terminal end of theCH1 domain of the Cκ/CH1 heterodimerization motif (FIG. 1). They havesurprisingly shown that said fusion allows the production of variousfunctional antigen binding formats differing in size and valence for thetargeted antigens. The fact that sdAbs fused via their N-terminal domainto the C-terminal domain of CH1 remain active is remarkable because thisN-terminal end is located at the vicinity of the antigen binding site.Major steric hindrance between the antigen and the heterodimerizationmotif might have occurred. However the inventors demonstrated thatfunctional antigen binding formats may be obtained.

Antigen-Binding Formats of the Invention

Accordingly, the present invention relates to an antigen-binding formatconsisting of:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a variable domain of an antibody and ii) by its C-terminal        end to the N-terminal end of a variable domain of an antibody        and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a variable domain of an antibody.

According to the invention, the antigen binding format of the inventionrepresents a heterodimeric format that reproduces the CL/CH1heterodimerization motif of a classic antibody and thus allows thecorrect folding of the antigen binding formats of the invention in acellular context. According to the invention, the CH1 constant domain ofthe first fusion protein and the CL constant domain of the second fusionprotein are therefore linked together via a disulfide bond.

According to the present invention, “antibody” or “immunoglobulin” havethe same meaning, and will be used equally in the present invention. Innatural antibodies, the two heavy chains are linked to each other bydisulfide bonds and each heavy chain is linked to a light chain by adisulfide bond. There are two types of light chain, lambda (λ) and kappa(κ). There are five main heavy chain classes (or isotypes) whichdetermine the functional activity of an antibody molecule: IgM, IgD,IgG, IgA and IgE. Each chain contains distinct sequence domains. Thelight chain includes two domains, a variable domain (VL) and a constantdomain (CL). The heavy chain includes four domains, a variable domain(VH) and three constant domains (CH1, CH2 and CH3, collectively referredto as CH). The variable regions of both light (VL) and heavy (VH) chainsdetermine binding recognition and specificity to the antigen. Theconstant region domains of the light (CL) and heavy (CH) chains conferimportant biological properties such as antibody chain association,secretion, transplacental mobility, complement binding, and binding toFc receptors. The Fv fragment is the N-terminal part of the Fab fragmentof an immunoglobulin consisting of the variable domains of one lightchain and one heavy chain. The specificity of the antibody resides inthe structural complementarity between the antibody combining site andthe antigenic determinant. Antibody combining sites are made up ofresidues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site. The CDR refers to amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native immunoglobulin binding site. The light and heavychain variable domains of an immunoglobulin have three CDRs, designatedL-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Anantigen-binding site, therefore, includes six CDRs, comprising the CDRset from each of a heavy and a light chain V region. FR refers to aminoacid sequences interposed between CDRs.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody molecule of a single amino acid composition, that is directedagainst a specific antigen and that is produced by a single clone of Bcells or hybridoma. Accordingly, the term “hybridoma” denotes a cell,which is obtained by subjecting a B cell prepared by immunizing ananimal with an antigen to cell fusion with a myeloma cell derived from amouse or the like which produces a desired monoclonal antibody having anantigen specificity.

In a particular embodiment, the CH1 and CL constant domains of theinvention are humanized constant domains, and more preferably full-humanCH1 and CL constant domains.

By “humanized”, it is meant mutated so that immunogenicity uponadministration in human patients is minor or nonexistent. Humanizing anantibody (e.g. a murine or Camelid antibody), according to the presentinvention, comprises a step of replacing one or more of the amino acidsof said antibody by their human counterpart as found in the humanconsensus sequence, without that antibody losing its typical character,i. e. the humanization does not significantly affect the antigen bindingcapacity of the resulting antibody.

In a particular embodiment, the CL domain is from a lambda (λ) or akappa (κ) light chain.

In a particular embodiment, the CH1 domain is from an IgG, such as IgG1,IgG2, IgG3, or IgG4. Alternatively, the CH1 domain is from an IgA, IgD,IgE or IgM.

In a particular embodiment, the variable domain is selected from thegroup consisting of VH domains, VL domains, or single domain antibodies(sdAbs).

The term “single domain antibody” (sdAb) or “VHH” refers to the singleheavy chain variable domain of antibodies of the type that can be foundin Camelid mammals which are naturally devoid of light chains. Such VHHare also called “nanobody®”. According to the invention, sdAb canparticularly be llama sdAb.

In a particular embodiment, the single domain antibody (VHH) domain ishumanized.

In a particular embodiment, the variable domain is a VH domain or asingle domain antibody (sdAb).

In a particular embodiment, the VH domain is a humanized VH domain, andmore preferably a fully human VH domain.

Typically, the variable domain may be directed against any antigen.

For example, the variable domain may be specific for an immune cellregulatory molecule such as CD3, CD4, CD8, CD25, CD28, CD26, CTLA-4,ICOS, or CD11a. Other suitable antigens include but are not limited tothose associated with immune cells including T cell-associatedmolecules, such as TCR/CD3 or CD2; NK cell-associated targets such asNKG2D, FcγRIIIa (CD16), CD38, CD44, CD56, or CD69;granulocyte-associated targets such as FcγRI (CD64), FcαRI (CD89), andCR3 (CD11b/CD18); monocyte/macrophage-associated targets (such as FcγRI(CD64), FcαRI (CD89), CD3 (CD11b/CD18), or mannose receptor; dendriticcell-associated targets such as FcγRI (CD64) or mannose receptor; anderythrocyte-associated targets such as CRI (CD35).

Alternatively, the variable domain according to the invention may bedirected against a cancer antigen. Known cancer antigens include,without limitation, c-erbB-2 (erbB-2 is also known as c-neu or HER-2),which is particularly associated with breast, ovarian, and colon tumorcells, as well as neuroblastoma, lung cancer, thyroid cancer, pancreaticcancer, prostate cancer, renal cancer and cancers of the digestivetract. Another class of cancer antigens is oncofetal proteins ofnonenzymatic function. These antigens are found in a variety ofneoplasms, and are often referred to as “tumor-associated antigens.”Carcinoembryonic antigen (CEA), and α-fetoprotein (AFP) are two examplesof such cancer antigens. AFP levels rise in patients with hepatocellularcarcinoma: 69% of patients with liver cancer express high levels of AFPin their serum. CEA is a serum glycoprotein of 200 kDa found inadenocarcinoma of colon, as well as cancers of the lung andgenitourinary tract. Yet another class of cancer antigens is thoseantigens unique to a particular tumor, referred to sometimes as “tumorspecific antigens,” such as heat shock proteins (e.g., hsp70 or hsp90proteins) from a particular type of tumor. Other targets include theMICA/B ligands of NKG2D. These molecules are expressed on many types oftumors, but not normally on healthy cells.

Additional specific examples of cancer antigens include epithelial celladhesion molecule (Ep-CAM/TACSTD1), mesothelin, tumor-associatedglycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7,cancer-associated viral vaccines (e.g., human papillomavirus antigens),prostate specific antigen (PSA, PSMA), RAGE (renal antigen), CAMEL(CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6,-C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, andSAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), cancer-associatedganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1,MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, and thelike (see also, e.g., Acres et al., Curr Opin Mol Ther 2004 February,6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 Oct. 8;1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003 July-August; 2(4Suppl 1):S161-8; and Ohshima et al., Int J Cancer. 2001 Jul. 1;93(1):91-6). Other exemplary cancer antigen targets include CA 195tumor-associated antigen-like antigen (see, e.g., U.S. Pat. No.5,324,822) and female urine squamous cell carcinoma-like antigens (see,e.g., U.S. Pat. No. 5,306,811), and the breast cell cancer antigensdescribed in U.S. Pat. No. 4,960,716.

The variable domain according to the invention may target proteinantigens, carbohydrate antigens, or glycosylated proteins. For example,the variable domain can target glycosylation groups of antigens that arepreferentially produced by transformed (neoplastic or cancerous) cells,infected cells, and the like (cells associated with other immunesystem-related disorders). In one aspect, the antigen is atumor-associated antigen. In an exemplary aspect, the antigen isO-acetylated-GD2 or glypican-3. In another particular aspect, theantigen is one of the Thomsen-Friedenreich (TF) antigens (TFAs).

The variable domain according to the invention can also exhibitspecificity for a cancer-associated protein. Such proteins can includeany protein associated with cancer progression. Examples of suchproteins include angiogenesis factors associated with tumor growth, suchas vascular endothelial growth factors (VEGFs), fibroblast growthfactors (FGFs), tissue factor (TF), epidermal growth factors (EGFs), andreceptors thereof; factors associated with tumor invasiveness; and otherreceptors associated with cancer progression (e.g., one of the HER1-HER4receptors).

Alternatively the variable domain according to the invention can bespecific for a virus, a bacteria or parasite associated target. Forexample, the variable domain may be specific for a virus-associatedtarget such as an HIV protein (e.g., gp120 or gp41), CMV or otherviruses, such as hepatitis C virus (HCV).

The variable domain according to the invention may also target albuminor FcRn to increase the half-life of the antigen binding formats of theinvention in the systemic circulation.

The variable domain according to the invention may alternatively targeta hapten, and more particularly low molecular weight hapten, and morepreferably a radiolabeled low molecular weight hapten. Molecular weighthaptens according to the invention may be selected from the groupconsisting of methotrexate, histamine succinyl glycine, DTPA (diethylenetriaminepentaacetic acid); DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid); andderivatives thereof (see, for example, U.S. Pat. Nos. 4,885,363;5,087,440; 5,155,215; 5,188,816; 5,219,553; 5,262,532; and 5,358,704;and D. Meyer et al., Invest. Radiol. 1990, 25: S53-55). In particularembodiment, the hapten is labeled with a radionuclide. For example,useful diagnostic radionuclides include, but are not limited to, ¹¹⁰In,¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁹Zr, ⁹⁴Tc, ¹⁵⁰Re,¹⁸⁸Re, or other gamma-, beta-, or positron-emitters. Particularly usefultherapeutic radionuclides include, but are not limited to ¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc,¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr,¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.

The constant domains and variable domains according to the invention canreadily be prepared by an ordinarily skilled artisan using routineexperimentation.

For example, the constant domains and variable domains according to theinvention may be from monoclonal antibodies. Monoclonal antibodiesdirected against antigens of interest can be produced by an animal(including, but not limited to, human, mouse, camelid, rat, rabbit,hamster, goat, horse, chicken, or turkey), chemically synthesized, orrecombinantly expressed. For example, monoclonal antibodies can beprepared and isolated using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture.Techniques for production and isolation include but are not limited tothe hybridoma technique originally described by Kohler and Milstein(1975); the human B-cell hybridoma technique (Cote et al., 1983); andthe EBV-hybridoma technique (Cole et al. 1985). Other known methods ofproducing transformed B cell lines that produce monoclonal antibodiesmay also be used. Monoclonal antibodies of the present invention may beproduced by recombinant DNA techniques, for example, produced by phagedisplay or by combinatorial methods. See, for example, U.S. Pat. No.5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO93/01288; WO 92/01047; WO 92/09690; or WO 90/02809. Monoclonalantibodies of the present invention can be purified by any method knownin the art for purification of an immunoglobulin molecule, for example,by chromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In addition,the antibodies can be fused to heterologous polypeptide sequencesdescribed herein or otherwise known in the art, to facilitatepurification. Finally, the genes encoding the constant domain orvariable domain according to the invention can be recovered from the DNAof relevant hybridomas or phages. In a particular embodiment, monoclonalantibodies are full-human antibodies or humanized antibodies.

sdAbs are usually generated by PCR cloning of the V-domain repertoirefrom blood, lymph node, or spleen cDNA obtained from immunized animalsinto a phage display vector, such as pHEN2. Antigen-specific sdAbs arecommonly selected by panning phage libraries on immobilized antigen,e.g., antigen coated onto the plastic surface of a test tube,biotinylated antigens immobilized on streptavidin beads, or membraneproteins expressed on the surface of cells. However, such sdAbs oftenshow lower affinities for their antigen than sdAbs derived from animalsthat have received several immunizations. The high affinity of sdAbsfrom immune libraries is attributed to the natural selection of variantsdAbs during clonal expansion of B-cells in the lymphoid organs ofimmunized animals. The affinity of sdAbs from non-immune libraries canoften be improved by mimicking this strategy in vitro, i.e., by sitedirected mutagenesis of the CDR regions and further rounds of panning onimmobilized antigen under conditions of increased stringency (highertemperature, high or low salt concentration, high or low pH, and lowantigen concentrations). sdAbs derived from camelid are readilyexpressed in and purified from the E. coli periplasm at much higherlevels than the corresponding domains of conventional antibodies. sdAbsgenerally display high solubility and stability and can also be readilyproduced in yeast, plant, and mammalian cells. For example, the “Hamerspatents” describe methods and techniques for generating VHH against anydesired target (see for example U.S. Pat. No. 5,800,988; U.S. Pat. No.5,874,541 and U.S. Pat. No. 6,015,695). The “Hamers patents” moreparticularly describe production of sdAbs in bacterial hosts such as E.coli (see for example U.S. Pat. No. 6,765,087) and in lower eukaryotichosts such as moulds (for example Aspergillus or Trichoderma) or inyeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia)(see for example U.S. Pat. No. 6,838,254). In a specific antibody, thesdAbs of the invention may be further humanized.

According to the invention, the CH1 and CL constant domains of thefusion proteins are fused directly at their N-terminal ends to theC-terminal end of a variable domain of an antibody.

As used herein, the term “directly” means that the (first or last) aminoacid at the terminal end (N or C-terminal end) of the constant domain isfused to the (first or last) amino acid at the terminal end (N orC-terminal end) of the variable domain.

In other words, in this embodiment, the first amino acid of theN-terminal end of said CH1 or CL constant domain is directly linked by acovalent bond to the last amino acid of the C-terminal end of saidvariable domain of an antibody.

According to the invention, the CH1 constant domain of the first fusionprotein is fused by its C-terminal end to the N-terminal end of avariable domain of an antibody either directly or via a spacer.

As used herein, the term “spacer” refers to a sequence of at least oneamino acid that links the constant domain with the variable domain ofthe invention. Such a spacer may be useful to prevent steric hindrances.Typically, said spacer is an amino acid sequence selected from the groupconsisting of AAA (SEQ ID NO:29) and DKT (SEQ ID NO:30). Preferably,said spacer is the sequence DKT naturally present at the C-terminal endof the human CH1 domain of antibodies.

Preferably, the antigen binding format according to the inventionconsists of:

-   -   a first fusion protein consisting of a CH1 domain of an antibody        fused directly by its N-terminal end to the C-terminal end of a        variable domain of an antibody, and fused via a spacer by its        C-terminal end to the N-terminal end of a variable domain of an        antibody, said spacer being preferably AAA or DKT and,    -   a second fusion protein consisting of a CL domain of an antibody        fused directly by its N-terminal end to the C-terminal end of a        variable domain of an antibody.

According to the invention, the antigen binding formats of the inventionhave 3 variable domains and can therefore be mono, bi, or tri specifictoward antigens of interest. Accordingly, every variable domains of theantigen binding format according to the invention has the ability tobind to an antigen by itself. The binding to the antigen is not as thesame as classically observed for an antibody binding to an antigen (theentity responsible for the binding to the antigen is the complex formedby the VL and CH variable domains) but approaches to the same asobserved for camelid mammals: a variable domain (VH, VL or VHH) can bindto an antigen by itself.

In a particular embodiment, the present invention relates to anantigen-binding format consisting of:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a variable domain of an antibody and ii) by its C-terminal        end to the N-terminal end of a variable domain of an antibody        and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a variable domain of an antibody.

with the exception of an antigen-binding format consisting of:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a VH variable domain of an antibody and ii) by its C-terminal        end to the N-terminal end of a variable domain of an antibody        and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a VL variable domain of an antibody    -   wherein the VH variable domain of the first fusion protein and        VL variable domain of the second fusion protein do not        constitute a unique antigen-binding site (i.e. the assembly of 2        fusion proteins does not constitute a fragment antigen-binding        (Fab fragment)).

In a particular embodiment, the present invention relates to anantigen-binding format consisting of:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a variable domain of an antibody and ii) by its C-terminal        end to the N-terminal end of a variable domain of an antibody        and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a variable domain of an antibody.

with the exception of an antigen-binding format consisting of:

-   -   a first fusion protein wherein the CH1 constant domain of an        antibody is fused i) by its N-terminal end to the C-terminal end        of a VH variable domain of an antibody and ii) by its C-terminal        end to the N-terminal end of a variable domain of an antibody        and,    -   a second fusion protein wherein the CL constant domain of an        antibody is fused by its N-terminal end to the C-terminal end of        a VL variable domain of an antibody

In some embodiments, the antigen binding format of the invention may bemonospecific when all variable domains are directed against the sameantigen. Moreover, the antigen binding format of the invention may bebispecific, when combination of variable domains makes them directedagainst two particular antigens (e.g. CEA and CD16). According to thisembodiment, said antigen binding format is bivalent; i.e. two of thethree variable domains are directed against the same antigen (e.g. CEAor CD16). Furthermore, the antigen binding format of the invention maybe trispecific, when combination of variable domains makes them directedagainst three particular antigens (e.g. CEA, CD3 and CD16 or CEA, CD16,human albumin or CEA, CD3, human albumin).

In a particular embodiment, the antigen binding format of the inventionis bispecific wherein two variable domains are specific for a cancerantigen (e.g. CEA) and the last variable domain is specific for animmune cell regulatory molecule (e.g. CD16).

In a particular embodiment, the antigen binding format of the inventionis trispecific wherein a first variable domain is specific for a cancerantigen (e.g. CEA or any cancer antigen), a second variable domain isspecific for a fist immune cell regulatory molecule (e.g. CD16), and athird variable domain is specific for a second immune cell regulatorymolecule (e.g. CD3). More particularly, the invention relates to anantigen binding format wherein a first variable domain is specific for acancer antigen, a second variable domain is specific for a naturalkiller cell molecule (e.g. CD16), and a third variable domain isspecific for a T cell molecule (e.g. CD3). provides the advantage tokill tumor cells more potently by provinding both NK cells and T cellsretargeting. Said binding antigen format also contributes to thestimulation of the adaptive immune system, because tumor killingT-lymphocytes could potentially be generated, and vaccination could beestablished (ie, the provision of long-term immune response and memorycells that could rapidly generate a new and efficient response in thecase of metastasis development).

In a particular embodiment, the antigen binding format of the inventionconsists of:

-   -   a first fusion protein consisting of a CH1 domain fused directly        by its N-terminal end to a single domain antibody specific for        CD16 and fused via a spacer by its C-terminal end to a single        domain antibody specific for carcinoembryonic antigen (CEA)        wherein said spacer is AAA or DKT; and    -   a second fusion protein consisting of a CL domain of a kappa (κ)        light chain fused directly by its N-terminal end with a single        domain antibody specific for carcinoembryonic antigen (CEA).

In a particular embodiment, an antigen binding format of the inventionis represented by format 514-515 or 1214-1215 in FIG. 1.

In a particular embodiment, the antigen binding format of the inventionis trispecific wherein the first variable domain is specific for acancer antigen (e.g. CEA), the second variable domain is specific for aimmune cell regulatory molecule (e.g. CD16), and the third variabledomain is specific for albumin. More particularly, the invention relatesto an antigen binding format wherein a first variable domain is specificfor a cancer antigen, a second variable domain is specific for a naturalkiller cell molecule (e.g. CD16), and a third variable domain isspecific for a albumin. The half life of the antigen binding format inthe systemic circulation may be thus increased without affecting itstumor cell killing effects.

Nucleic Acids, Vectors and Recombinant Host Cells of the Invention

A further object of the present invention relates to a nucleic acidmolecule encoding for an antigen binding format according to theinvention.

As used herein, a sequence “encoding” an expression product, such as aRNA, polypeptide, protein, or enzyme, is a nucleotide sequence that,when expressed, results in the production of that RNA, polypeptide,protein, or enzyme, i.e., the nucleotide sequence encodes an amino acidsequence for that polypeptide, protein or enzyme. A coding sequence fora protein may include a start codon (usually ATG) and a stop codon.

These nucleic acid molecules can be obtained by conventional methodswell known to those skilled in the art.

Typically, said nucleic acid is a DNA or RNA molecule, which may beincluded in a suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or viral vector.

So, a further object of the present invention relates to a vector and anexpression cassette in which a nucleic acid molecule encoding for anantigen binding format of the invention is associated with suitableelements for controlling transcription (in particular promoter, enhancerand, optionally, terminator) and, optionally translation, and also therecombinant vectors into which a nucleic acid molecule in accordancewith the invention is inserted. These recombinant vectors may, forexample, be cloning vectors, or expression vectors.

As used herein, the terms “vector”, “cloning vector” and “expressionvector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreigngene) can be introduced into a host cell, so as to transform the hostand promote expression (e.g. transcription and translation) of theintroduced sequence.

According to the invention, the vector is a bicistronic vector thatincludes two nucleic acid molecules, each one encoding for a fusionprotein of the antigen binding format.

Any expression vector for animal cell can be used. Examples of suitablevectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T etal. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981),pSG1 beta d2-4-(Miyaji H et al. 1990) and the like.

Other examples of plasmids include replicating plasmids comprising anorigin of replication, or integrative plasmids, such as for instancepUC, pcDNA, pBR, and the like.

Other examples of viral vectors include adenoviral, retroviral, herpesvirus and AAV vectors. Such recombinant viruses may be produced bytechniques known in the art, such as by transfecting packaging cells orby transient transfection with helper plasmids or viruses. Typicalexamples of virus packaging cells include PA317 cells, PsiCRIP cells,GPenv+ cells, 293 cells, etc. Detailed protocols for producing suchreplication-defective recombinant viruses may be found for instance inWO 95/14785, WO 96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478.

Examples of promoters and enhancers used in the expression vector foranimal cell include early promoter and enhancer of SV40 (Mizukami T. etal. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus(Kuwana Y et al. 1987), promoter (Mason J O et al. 1985) and enhancer(Gillies S D et al. 1983) of immunoglobulin H chain and the like.

The invention also includes gene delivery systems comprising a nucleicacid molecule of the invention, which can be used in gene therapy invivo or ex vivo. This includes for instance viral transfer vectors suchas those derived from retrovirus, adenovirus, adeno associated virus,lentivirus, which are conventionally used in gene therapy. This alsoincludes gene delivery systems comprising a nucleic acid molecule of theinvention and a non-viral gene delivery vehicle. Examples of non viralgene delivery vehicles include liposomes and polymers such aspolyethylenimines, cyclodextrins, histidine/lysine (HK) polymers, etc.

A subject of the present invention is also a prokaryotic or eukaryotichost cell genetically transformed with at least one nucleic acidmolecule or vector according to the invention (preferably a bicistronicvector as above described).

The term “transformation” means the introduction of a “foreign” (i.e.extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. A host cell that receives and expressesintroduced DNA or RNA has been “transformed”.

In a particular embodiment, for expressing and producing antigen bindingformats of the invention, prokaryotic cells, in particular E. colicells, will be chosen. Actually, according to the invention, it is notmandatory to produce the antigen binding format of the invention in aeukaryotic context that will favour post-translational modifications(e.g. glycosylation). Furthermore, prokaryotic cells have the advantagesto produce protein in large amounts. If a eukaryotic context is needed,yeasts (e.g. saccharomyces strains) may be particularly suitable sincethey allow production of large amounts of proteins. Otherwise, typicaleukaryotic cell lines such as CHO, BHK-21, COS-7, C127, PER.C6, YB2/0 orHEK293 could be used, for their ability to process to the rightpost-translational modifications of the antigen binding format of theinvention.

Accordingly, a further aspect of the invention relates to a host cellcomprising a nucleic acid molecule encoding for an antigen bindingformat according to the invention or a vector according to theinvention.

The construction of expression vectors in accordance with the invention,and the transformation of the host cells can be carried out usingconventional molecular biology techniques. The antigen binding formatsof the invention, can, for example, be obtained by culturing geneticallytransformed cells in accordance with the invention and recovering theantigen binding format expressed by said cell, from the culture. Theymay then, if necessary, be purified by conventional procedures, known inthemselves to those skilled in the art, for example by fractionalprecipitation, in particular ammonium sulfate precipitation,electrophoresis, gel filtration, affinity chromatography, etc. . . . Inparticular, conventional methods for preparing and purifying recombinantproteins may be used for producing the proteins in accordance with theinvention.

A further aspect of the invention relates to a method for producing anantigen binding format of the invention comprising the step consistingof: (i) culturing a transformed host cell according to the inventionunder conditions suitable to allow expression of said antigen bindingformat; and (ii) recovering the expressed antigen binding format.

Therapeutic Methods and Uses of the Invention

The present invention provides methods and compositions (such aspharmaceutical compositions) for treating cancer or infectious diseases.Antigen binding formats of the invention are indeed particularlysuitable for the treatment of diseases such as cancer or infectiousdiseases.

Therefore, a further object of the invention relates to an antigenbinding format of the invention for use as a medicament.

More particularly, an aspect of the invention relates to an antigenbinding format of the invention for use in the treatment of cancer orinfectious diseases.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition.

As used herein, the term “infectious disease” is intended to encompassany disease which results from an infection mediated by a virus, abacteria or a parasite. Therefore the term includes but is not limitedto infection with virus such as human immunodeficiency virus, HepatitisB virus, hepatitis C virus, with parasites such as Plasmodium Falciparum(causative agent for Malaria), or with bacteria such as mycobacteriumtuberculosis.

As used herein, the term “cancer” is intended to encompass primary andmetastatic solid tumors, including carcinomas of breast, colon, rectum,lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,gallbladder and bile ducts, small intestine, kidney, bladder,urothelium, female genital tract, (including cervix, uterus, and ovariesas well as choriocarcinoma and gestational trophoblastic disease), malegenital tract (including prostate, seminal vesicles, testes and germcell tumors), endocrine glands (including the thyroid, adrenal, andpituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma) and tumors of the brain, nerves, eyes, such as astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas, and meningiomas. The term cancer also relates to tumorsarising from hematopoietic malignancies such as leukemias as well bothHodgkin's and non-Hodgkin's lymphomas.

A further aspect of the invention relates to a method for treatingcancer or an infectious disease, comprising administering to a subjectin need thereof an amount of antigen binding format according to theinvention.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably, a subject according to theinvention is a human.

The antigen binding format of the invention may be administered in theform of a pharmaceutical composition, as defined below.

Preferably, the antigen binding formats of the invention areadministered in a therapeutically effective amount.

The term “therapeutically effective amount” means a sufficient amount ofthe active ingredients of the invention to treat cancer or infectiousdisease at a reasonable benefit/risk ratio applicable to any medicaltreatment.

It will be understood that the total daily usage of the antigen bindingformats of the invention will be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed, the age, body weight, general health, sexand diet of the subject; the time of administration, route ofadministration, and rate of excretion of the specific antigen bindingformats employed; the duration of the treatment; drugs used incombination or coincidental with the specific antigen binding formatsemployed; and like factors well known in the medical arts. For example,it is well within the skill of the art to start doses of the antigenbinding formats at levels lower than those required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

An approach to cancer therapy and diagnosis may also involve the use ofa bispecific antigen binding format according to the invention having atleast one arm that specifically binds a cancer antigen and at least oneother arm that specifically binds a low molecular weight hapten. In thismethodology, the antigen binding format of the invention is administeredand allowed to target the cancer antigen and therefore the tumour. Sometime later, a radiolabeled low molecular weight hapten is given, whichbeing recognized by the second specificity of the antigen bindingformat, also localizes to the tumour.

In another particular embodiment, the antigen binding format accordingto the invention may act as a ligand for a cell receptor or channel.Therefore the antigen binding format according to the invention mayrepresent an agonist, a partial agonist or an antagonist for a receptor.

Diagnostic Methods and Uses of the Invention

Antigen binding formats of the invention may also be particularlysuitable for diagnosing or monitoring a disease. Said disease may be anydisease and may be selected for example from the group consisting ofcancers and infectious diseases.

A further aspect of the invention relates to the use of an antigenbinding format of the invention for diagnosing or monitoring a diseasethat may be selected from the group consisting of cancers and infectiousdiseases.

In a particular embodiment, antigen binding formats of the invention maybe labelled with a detectable molecule or substance, such as afluorescent molecule, a radioactive molecule or any others labels knownin the art. Labels are known in the art to generally provide (eitherdirectly or indirectly) a signal.

As used herein, the term “labelled”, with regard to the antibody, isintended to encompass direct labelling of the antibody by coupling(i.e., physically linking) a detectable substance, such as a radioactiveagent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) orphycoerythrin (PE) or indocyanine (Cy5)) to the antibody, as well asindirect labelling of the antibody by reactivity with a detectablesubstance.

An antigen binding format of the invention may be labelled with aradioactive molecule by any method known to the art. For exampleradioactive molecules include but are not limited to radioactive atomsfor scintigraphic studies such as I123, I124, In111, Re186, Re188.Antigen binding formats of the invention may also be labelled with aspin label for nuclear magnetic resonance (NMR) imaging (also known asmagnetic resonance imaging, mri), such as iodine-123, iodine-131,indium-I11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

A “biological sample” encompasses a variety of sample types obtainedfrom a subject and can be used in a diagnostic or monitoring assay.Biological samples include but are not limited to blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. For example, biological samples include cells obtained from atissue sample collected from an individual suspected of having a cancerdisease. Therefore, biological samples encompass clinical samples, cellsin culture, cell supernatants, cell lysates, serum, plasma, biologicalfluid, and tissue samples.

Antigen binding formats of the invention may be useful for staging ofcancer diseases (e.g., in radioimaging).

Pharmaceutical Compositions and Kits of the Invention

The invention also relates to pharmaceutical compositions comprisingantigen binding formats of the invention.

Therefore, antigen binding formats of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce any adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment.

To prepare pharmaceutical compositions, an effective amount of theantigen binding format may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

An antigen binding format of the invention can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the requiredamount of the active compounds in the appropriate solvent withvarious/several of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and the otherrequired ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The antigen binding formats of the invention may be formulated within atherapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about0.001 to 1.0 milligrams, or about 0.01 to 1.0 milligrams, or about 0.1to 1.0 milligrams or even about 10 milligrams per dose or so. Multipledoses can also be administered.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; time release capsules; and any other form currentlyused.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of antigen binding formats into hostcells. The formation and use of liposomes and/or nanoparticles are knownto those of skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) are generally designedusing polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beeasily made.

Liposomes are formed from phospholipids that once dispersed in anaqueous medium spontaneously form multilamellar concentric bilayervesicles (also termed multilamellar vesicles (MLVs)). MLVs generallyhave diameters ranging from 25 nm to 4 μm. Sonication of MLVs results inthe formation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations.

The invention also provides kits comprising at least one antigen bindingformat of the invention. Kits containing antigen binding formats of theinvention find use in therapeutic methods or diagnostic assays.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Representation of various bispecific antibody formats.

FIG. 2: Flow cytometry experiments demonstrating a higher apparentaffinity of bivalent (bbsAb) vs. monovalent (bsFab) bispecificantibodies. CEA: MC38-CEA cells CD16: Jurkat-CD16 cells.

FIG. 3: In vitro cytotoxicity assays performed by flow cytometry usinghuman NK cells and bispecific antibodies

EXAMPLE Material & Methods 1/ Construction of the New Formats

p501 (SEQ ID NO:9): Insertion of anti-CEA sdAb 17 gene (Behar,G.,Chames,P., Teulon,I., Cornillon,A., Alshoukr,F., Roquet,F., Pugniere,M.,Teillaud,J.-L., Gruaz-Guyon,A., Pelegrin,A. and Baty,D Llama singledomain antibodies directed against non-conventional epitopes oftumor-associated carcinoembryonic antigen (CEA) absent from non-specificcross-reacting antigen (NCA). FEBS J, 2009, 276, 3881-93) of pHen1-CEA17(SEQ ID NO:10) into p1 (SEQ ID NO:11) at the C-terminal end of CH1domain (Not I site):

PCR: 5 ng pHen1-CEA17 (SEQ ID NO:10), 0.5 U Taq Polymerase Deep-vent(New England Biolabs), 10 μM of primer 5′ Not VHH and primer 3′ Not VHHrev, 5 μL Tp 10X, 1 μl MgSO₄ 1 mM, 4 μl dNTP 2.5 mM, H₂O up to 50 μl,94° C. 3 min, 94° C. 45 sec, 60° C. 45 sec, 72° C. 45 sec×35 cycles, 72°C. 10 min.

Primer 5′ Not VHH Not SEQ ID NO 1:CCACGATTCTGCGGCCGCAGAGGTGCAGCTGGTGGAGTCTGG Primer 3′ Not VHH revSEQ ID NO 2: TTTTTGTTCTGCGGCCGCTGAGGAGACGGTGACCTGGG

The PCR fragment was purified on 2% agarose gel using NucleoSpin ExtractII kit (Macherey-Nagel) and eluted in 50 μL of buffer NE.

Digestion of p1 (SEQ ID NO:11) (5 μg) by Not I (50 U), 30 μL buffer 310X, 30 μL BSA 10 mg/ml, H₂O up to 300 μl 1 h at 37° C. followed bydephosphorylation using 10 U of CIP (Biolabs), 30 min at 37° C. CIP wasinactivated using 9 μl EDTA 0.5 M 1 h at 65° C., phenol-treated, andprecipitated 16 h at −20° C. using 1 volume NaOAc 0.3M and 3 volumesethanol 96%, followed by centrifugation 10 min at 16 000 g, washing ofthe pellet using 750 μl ethanol 70%. The dried pellet was resuspended in20 μl H₂O.

PCR fragment (20 μl) was digested using NotI (100 U) (30 μl buffer 310X, 30 μl BSA 10 mg/ml, H₂O up to 300 μl 1 h at 37° C.). After phenoltreatment, the digested PCR fragment was precipitated 16 h at −20° C.using 1 volume NaOAc 0.3 M and 3 volumes ethanol 96%, followed bycentrifugation 10 min at 16 000g, washing of the pellet using 750 μlethanol 70%. The dried pellet was resuspended in 20 μL H₂O.

Ligation: 120 ng of vector p1 (SEQ ID NO:11) and 60 ng of PCR fragmentusing 10 U of ligase (Biolabs), 1 μl of buffer 10X, H₂O up to 10 μl, 1 hat RT, 10 min at 65° C.

Three μl of the ligation was electroporated into 40 μl ofelectrocompetent TG1 cell. Cells were resuspended in 1 ml SOC, 1 h at37° C., shaked at 180 rpm, and plate on LB/ampicillin dishes.

Two 2 sets of colony-PCRs were performed on 48 colonies to check theligation event.

PCR1: Primer 5′ M13 Reverse SEQ ID NO 3: CAGGAAACAGCTATGAC and Primer 3′Hind 111 + 40 SEQ ID NO 4: GCTGAAAATCTTCTCTCATCCG PCR2: Primer 5′ FlagSEQ ID NO 5: GCAGGTGATTACAAAGACGATG and Primer 3′ Not VHH rev(SEQ ID NO 2)

(0.25 U Dynazyme II (Finnzyme), 2.5 μL buffer 10X, 10 mM dNTP, H₂O up to25 μL, 94° C. 3 min, 94° C. 45 sec, 60° C. 45 sec, 72° C. 45 sec×35cycles, 72° C. 10 min).

Four positive clones were assayed for expression: induction 12 h at 30°C., IPTG 100 μM, loading of cell lysate on SDS-PAGE 15%, and westernblot using 9E10-HRP diluted 1/100).

The sequences were checked (Millegen).

p514 (SEQ ID NO:12): Insertion of the NheI/HindIII fragment from p501(SEQ ID NO:9) into p14 (SEQ ID NO:13):

10 μg of each vector was digested in 10 μl buffer 2 10X, 10 μl of BSA(10 mg/ml) 20 U NheI, 20 U HindIII, H₂O up to 100 μl, 1 h at 37° C. p14(SEQ ID NO:13) was dephosphorylated and resuspended in TE (see above).

Fragments were gel purified as above using NucleoSpin Extract II(Macherey-Nagel) and eluted using 25 μl H₂O. Fragments were ligated andligation was electroporated as above.

Colony-PCR was performed using primers 5′ sigpelbfor (SEQ ID NO 6:TACCTATTGCCTACGGCAGCC) and 3′ HindIII+40 (SEQ ID NO 4), 0.25 U DynazymeII (Finnzyme), 2.5 μl buffer 10X, 4 μl dNTP 2.5 mM, H₂O up to 25 μl, 94°C. 3 min, 94° C. 1 min 30 sec, 60° C. 1 min 30 sec, 72° C. 1 min 30sec×35 cycles, 72° C. 10 min. Four positive clones were assayed forexpression as above, and sequences were checked (Millegen)

p515 (SEQ ID NO:14): Insertion of the NheI/HindIII fragment from p501(SEQ ID NO:9) into p15 (SEQ ID NO:15):

As for p514

p1201 (SEQ ID NO:16): Mutation from AAA to DKT spacer on p501 (SEQ IDNO:9):

Quikchange on p501 (SEQ ID NO:9) using 125 ng of each primer, 5 μl ofbuffer 10X, 4 μl dNTP, 2.5 U Pfu Ultra (Stratagene), H₂O up to 50 μl,95° C. 1 min, 95° C. 30 sec, 55° C. 1 min, 68° C. 7 min,×18 cycles, add40 U Dpn I

Primer 5′ 1201 for SEQ ID NO: 7:GTTGAGCCCAAATCTTGTGACAAAACTGAGGTGCAGCTGGTGG Primer 3′ 1201revSEQ ID NO 14: CCACCAGCTGCACCTCAGTTTTGTCACAAGATTTGGGCTCAAC

Transformation was performed by electroporation into DH5α using 2 μl ofQuikchange product.

Clones were tested for expression as above and sequences were checked.

p1214 (SEQ ID NO:17): Insertion of Nhe I/Hind III fragment from p1201(SEQ ID NO:16) into p14 (SEQ ID NO:13):

Digest 1 μg of p14 (SEQ ID NO:13) and 15 μg of p1201 (SEQ ID NO:16) byNheI 20 U for p14 (SEQ ID NO:8) and 60 U for p1201 (SEQ ID NO:16) andHindIII 20 U for p14 and 60 U for p1201 using buffer 2 1X final H₂O upto 50 μl, 2 h at 37° C., dephosphorylate vector 14 by adding 10 U ofCIP, 30 min at 37° C., add 1.5 μl EDTA 0.5M, followed by phenolextraction, ethanol precipitate and resuspend in 20 μl H₂O.

Gel-purify fragment from p1201 (SEQ ID NO:16) using NucleoSpin ExtractII.

Ligation using various vector ratios: insert, using 10 μl Buffer 2X, 3 UT4 DNA ligase (Promega), H₂O up to 50 μl, 15 min RT, 15 min at 65° C.,ethanol precipitate and resuspend in 4 μl H₂O

Electroporate 40 μl of DH5α using 4 μl of ligation, plate on LB/Ampi 100μg/ml petri dishes

Clones were tested for expression as above and sequences were checked.

p1215 (SEQ ID NO:18): as for p1214 (SEQ ID NO:17) but using p15 (SEQ IDNO:15):

2/ Production and Purification of Abs 2.1 Production in E. Coli

The E. coli K12 strain DH5α was used for the Ab production. First, asingle colony containing the plasmid of interest was inoculated in 50 mlof LB medium supplemented with ampicillin 100 μg/ml and 2% glucose. Thebacteria were grown at 30° C. and shaken at 205 rpm overnight. Fourhundred ml of LB medium supplemented with ampicillin 100 μg/ml wasinoculated with the previous culture to obtain an OD_(600 nm) of 0.1.The bacteria were grown at 30° C. for 2 h 30, then at 20° C. to anOD_(600 nm) of 0.8-1. The production of Abs was induced by addition of0.1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) for 60 h at 20° C.

2.2 Extraction of the Soluble Fraction of the Periplasm

Cells were harvested by centrifugation at 1860 g for 30 min at 4° C. Thepellet was thoroughly resuspended in 4 ml of cold TES buffer pH 8.0 (0.2M Tris HCl pH 8, 0.5 mM EDTA, 0.5 M Sucrose) before adding 1.6 mg offreshly made lysosyme solution. The cells were then submitted to anosmotic shock by addition of 8 ml of cold TES buffer and 8 ml of coldwater. After an incubation of 30 min on ice, 250 μg of DNAse I (Roche)and 250 μl of 1 M MgCl₂ were added and the mixture was further incubated30 min at room temperature. The mixture was centrifuged at 1860 g for 1h at 4° C. After addition of one tablet of a protease inhibitorcocktail, Complete EDTA-free (Roche) into 50 ml of supernatant, thesupernatant was dialyzed for 16 h at 4° C. against 50 mM sodium acetatebuffer pH 7.0 containing 0.1 M NaCl.

2.3 Purification of Abs Purification by Cobalt Affinity Chromatography

After filtration on 0.2 μm filter (Millipore), the supernatant wasloaded on a 2 ml BD Talon™ Metal Affinity column (BD BiosciencesClontech) previously equilibrated in a 50 mM sodium acetate buffer pH7.0 containing 0.1 M NaCl. The column was washed with 5 volumes of 50 mMsodium acetate buffer pH 7.0 containing 1 M NaCl then with 5 volumes of50 mM sodium acetate buffer pH 7.0 containing 0.1 M NaCl. Elution wasperformed by a linear imidazol gradient (0 to 250 mM). The elutionprofile was followed spectrophotometrically at 280 nm. The purifiedfractions were analyzed by SDS PAGE 12% stained with Coomassie blue andwestern blotting using anti cmyc-HRP antibody (1 μg/ml, Santa CruzBiotechnologies) and anti-flag-HRP antibody (0.5 μg/ml, Sigma).

Purification by Affinity Chromatography on Protein G

The fractions containing Abs were pooled, dialyzed against PBS buffer(137 mM NaCl, 2.7 mM KCl, 1.2 mM Na2HPO4, 1.76 mM KH2PO4 pH 7.4) andloaded onto a 1 ml Hi-Trap Protein G column (Amersham Biosciences).After washing the column with 5 volumes of PBS, the antibodies wereeluted with a 0.1 M glycine HCl buffer pH 2.7 and neutralized byaddition of 1/10 volume of 1 M Tris-HCl buffer pH 9.0. The elutionprofile was followed spectrophotometrically at 280 nm. Purification ofAbs was followed by SDS PAGE 12% stained with Coomassie blue and westernblotting. Fractions of interest were pooled, washed in PBS andconcentrated on Vivaspin devices (cut off 5 kDa, Millipore).

Ab concentrations were determined by the colorimetric method of Lowryusing the Biorad protein assay kit. Abs were diluted with one volume ofglycerol and stored at −20° C.

3/ Flow Cytometry Experiments

Binding of bsAb to CEA was assessed using MC38-CEA cells. Binding ofantibodies to CD16A was assessed using Jurkat-CD16A cells. Cells (5×10⁵cells/well) were distributed in a V-bottom 96 well microplate andincubated with various concentrations of antibodies (0.82 to 200 nM) for1 h at 4° C. All cell and antibody dilutions were performed in PBS 1%BSA. Bound antibodies were stained by monoclonal mouse anti c-mycantibody 9E10 (4 μg/ml, (Santa Cruz Biotechnology) for CEA binding ormouse anti-flag M2 antibody (1 μg/ml, Sigma) for CD16A binding, followedby F(ab′)2 goat anti-mouse-FITC antibody (7 μg/mL, Beckman Coulter).After several washes with PBS 1% BSA, labelled cells were gentlyresuspended in 200 μl PBS BSA 1% and analyzed by flow cytometry on aFACS Calibur cytometer (BD Biosciences). The results were analyzed withCellQuest Pro (BD Biosciences) or Flowjo (Treestart Inc.) softwares.

4/ In Vitro Cytotoxicity Assays 4.1 Isolation of PBMCs (Peripheral BloodMononuclear Cells)

A blood pack (400 ml) from a healthy donor was recovered in the EFS(Etablissement français du sang) Marseille, France. The blood wasdiluted in half in PBS 1% FCS and distributed in tubes of bloodseparation (PAA) previously filled with gradient separation (LSM 1077Lymphocyte, PAA). The tubes were centrifuged 40 min at 400 g at roomtemperature with no acceleration and no brake. The opaque ring at theinterface between plasma and gradient separation containing PBMCs wasrecovered. The cells were then washed twice in PBS 1% FCS andcentrifuged 20 min at 150 g at room temperature. The cells were thencounted on Malassez cell and either resuspended in FCS 20% DMSO andfrozen in nitrogen or used for further assays.

4.2 NK Cells Purification

The selection of NK (natural killer) cells was made by negativedepletion using the NK cell isolation kit (Milteny Biotec) from PBMCsfrom healthy donors according to manufactor's indications.

One hundred million of previously isolated PBMC were resuspended in 400μl of cold buffer (PBS 0.5% BSA 2 mM EDTA). The cells were incubatedwith 100 μl of a cocktail of antibodies: biotin NK cell antibodycocktail, 10 min on ice. After addition of 300 μL of cold buffer, thecells were incubated with 200 μl of magnetic beads: NK cell cocktailmicrobeads, 15 min on ice. The cells were then washed with 10 ml ofbuffer and then resuspended in 500 μl of buffer. The cell suspension wasloaded onto the MACS LS column placed in a magnetic field of a MACSeparator and washed with 9 ml of buffer. The unlabelled NK cells werecollected, counted and resuspended in culture medium RPMI 10% FCS.

4.3 Cytotoxicity Assay by Flow Cytometry

The cytotoxicity assay by flow cytometry quantifies the lysis of CEApositive target cells induced by CD16 + cells such as NK cells fromhealthy donors under the action of bsAb.

4.4 Labelling of Target Cells with CFSE

Five millions of CEA+ target cells (MC38-CEA) were washed twice in PBS1% BSA before addition of CFSE (Carboxyfluorescein succinimidyl ester,)at a final concentration of 5 μM in 1 ml of PBS 1% BSA for 10 min inwater bath at 37° C. The reaction was then stopped by adding an excessof cold PBS 1% BSA. Cells were washed twice in culture medium RPMI 10%FCS.

CFSE labeled target cells (20 000 cells in 100 μL per well) wereincubated with various concentrations of antibodies (1000 pM to 0.01pM). Each point was made in triplicate. NK cells freshly isolated fromPBMC were then added at a effector/target ratio of 10/1. The plate wascentrifuged at 560 g for 30 s and incubated at 37° C. for 12 h.

Cells were recovered and washed twice in PBS 1% BSA and incubated for 5min with 100 μL of 2 nM To-pro3. After addition of 100 μL of PBS 1% BSA,the samples were analyzed by flow cytometry (FACS Calibur, BDBiosciences). Target cell lysis was subsequently measured by release ofintracellular label by a scintillation counter or spectrophotometry.Dead target cells were identified as CFSE+/To-pro 3+ cells.

% lysis=(% Target+NK+Ab−% Target+NK)/(100−% Target+NK)×100.

Results

The modularity of single domain antibodies combined to the use of thehuman heterodimerization motif constituted by the CH1/Cκ domains allowsthe efficient generation of multivalent and/or multispecific recombinantantibodies. We have previously demonstrated the possibility to produceactive bispecific monovalent constructs named bsAb (see FIG. 1 andWO/2006/064136) allowing the efficient retargeting and activation ofeffector cells such as human NK cells and macrophages toward tumorcells, leading to their lysis.

In these constructs, the sdAbs were linked to the N-terminal extremitiesof CH1 and Cκ domains by their C-terminal extremities. In this work, wewanted to establish the possibility to link the N-terminal end of thesdAb to the the C-terminus of the CH1 domain to create multivalentbispecific molecules. Several molecules corresponding to the addition ofone anti-CEA domain to the original bsAb format were thus constructed.The N-terminus of sdAbs is located at the tip of the domain, i.e. closeto the antigen binding interface. To avoid possible steric clashes, theeffect of the addition of small spacers (3 residues, AAA or DKT) wasinvestigated. A representation of these various new formats is shown inFIG. 1.

All these molecules were produced in the periplasm of E. coli to allow aproper disulfide bond formation within each Ig domain and purified afterperiplasm extraction using a common two-steps procedure, i.e. metalaffinity chromatography followed by protein G purification.

Two of the new molecules, i.e. 1214 (SEQ ID NO:25 and SEQ ID NO:26) and1215 (SEQ ID NO:27 and SEQ ID NO:28) were compared with their monovalentparent molecules (14 and 15) chosen to demonstrate an avidity effect dueto the addition of an extra anti-CEA domain, using flow cytometry onCEA+ target cells.

FIG. 2 clearly shows that bivalent constructs 1214 and 1215 yieldedhigher signals than their parent molecules, especially at lowconcentration. Altogether, these results demonstrate that single domainadded at the C-terminus of CH1 can access and efficiently bind theirantigen displayed at the cell surface.

The ability of these new molecules to retarget effector cells towardCEA+ target cells was demonstrated using a flow cytometry based in vitrocytotoxicity assay. CEA+ target cells were fluorescently labelled usingCFSE and mixed to human NK cells at an effector: target ratio of 10:1 inthe presence of various concentration of monovalent (14 and 15) orbivalent (1214 and 1215) bispecific molecules. As shown in FIG. 3, allmolecules displayed a similar activity in this assay with EC₅₀ values inthe pM range.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1-10. (canceled)
 11. A nucleic acid molecule encoding for an antigenbinding format comprising: a first fusion protein wherein a CH1 constantdomain of an antibody is fused i) by its N-terminal end to a C-terminalend of a first single domain antibody and ii) by its C-terminal end toan N-terminal end of a second single domain antibody, wherein theC-terminal end of the CH1 constant domain includes DKT which is fuseddirectly to the N-terminal end of the second single domain antibody, anda second fusion protein wherein a CL constant domain of an antibody isfused by its N-terminal end to a C-terminal end of a third single domainantibody, wherein said first, second and third single domain antibodymay be the same or different, and wherein said CH1 constant domain andsaid CL constant domain are linked via a disulfide bond.
 12. A vectorcomprising a nucleic acid molecule encoding for an antigen bindingformat comprising: a first fusion protein wherein a CH1 constant domainof an antibody is fused i) by its N-terminal end to a C-terminal end ofa first single domain antibody and ii) by its C-terminal end to anN-terminal end of a second single domain antibody, wherein theC-terminal end of the CH1 constant domain includes DKT which is fuseddirectly to the N-terminal end of the second single domain antibody, anda second fusion protein wherein a CL constant domain of an antibody isfused by its N-terminal end to a C-terminal end of a third single domainantibody, wherein said first, second and third single domain antibodymay be the same or different, and wherein said CH1 constant domain andsaid CL constant domain are linked via a disulfide bond.
 13. Aprokaryotic or eukaryotic host cell genetically transformed with atleast one nucleic acid molecule encoding for an antigen binding formatcomprising: a first fusion protein wherein a CH1 constant domain of anantibody is fused i) by its N-terminal end to a C-terminal end of afirst single domain antibody and ii) by its C-terminal end to anN-terminal end of a second single domain antibody, wherein theC-terminal end of the CH1 constant domain includes DKT which is fuseddirectly to the N-terminal end of the second single domain antibody, anda second fusion protein wherein a CL constant domain of an antibody isfused by its N-terminal end to a C-terminal end of a third single domainantibody, wherein said first, second and third single domain antibodymay be the same or different, and wherein said CH1 constant domain andsaid CL constant domain are linked via a disulfide bond. 14-16.(canceled)
 17. The prokaryotic or eukaryotic host cell of claim 13,wherein said at least one nucleic acid molecule is present in a vector.